Recent innovations in type 2 diabetes therapeutics have focused on gut-derived incretin hormones that have distinct effects on insulin secretion, satiety, and body weight. One such hormone, glucagon-like peptide-1 (GLP-1), is released post-prandially from the intestine and potentiates glucose-dependent insulin secretion from pancreatic beta cells (among many other activities) via its family B G protein-coupled receptor (GPCR). GPCRs in general been highly successfully drugged with small molecules. The GLP-1 receptor (GLP1R), however, has eluded successful small molecule targeting likely because its large, complex orthosteric binding sites. Allosteric modulation (potentiation or suppression of activity generated by ligand in the orthosteric site by a small molecule that binds elsewhere or induces activity on its own), was therefore, an attractive mode of targeting the GLP1R. We have completed a high-throughput screen and initial structure-activity work in which ~200 compounds with unique modes of pharmacological activity, including positive allosteric modulation (PAM) and allosteric agonists of the GLP1R have been identified. Here we will employ a multi-dimensional iterative analog library synthesis approach, to optimize lead compound(s) for properties enabling in vivo use as a tool to dissect the role of unique modes of GLP-1R modulation in health and disease. We will develop both peripherally restricted as well as CNS-penetrant probes in order to address outstanding biological questions in vivo. Libraries will be tested in a suite of in vitro pharmacology assays to assess potency, efficacy, selectivity and these data will be utilized to triage for further medicinal chemistry and DMPK efforts. Those compounds possessing chemical and pharmacological merit will be advanced to in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies using contemporary methodology to identify, optimize, and formulate compounds with favorable drug-like properties (+/- CNS penetrance) for use in in vivo studies. In order to test the physiological significance of either peripherally restricted or centrally penetrant positive GLP1R PAMs we will utilize variations of state-of-the-art in vivo glucose homeostasis methods involving glucose and insulin clamping in the presence and absence of varying concentrations of peptide GLP1R agonist. We will also test effects, particularly of CNS penetrant compounds, on behaviors related to CNS GLP1R signaling such as feeding and neuroprotection. While GLP-1 based therapeutics have clear efficacy in diabetes and weight control, significant liabilities exist. We hypothesize that fine-tuning GLP1R signaling via PAM offers the potential to optimize therapeutic outcomes in diabetes, obesity, and other disorders. This approach may capitalize on native GLP-1 secretion, or may be coupled to combination therapy with other GLP-1 targeted therapeutics. The proposed studies will generate the tools, and initial proof-of-concept that such an approach may offer advantages.